Adrenocorticotrophin-gelatin preparation



Patented Feb. 16, 1954 OFFICE ADRENOCORTICOTROPHIN- GELATIN PREPARATIONRobert E. Thompson,

to Armour and Com poration of Illinois Cos Cob, Conn, assignor pany,Chicago, 111., a cor- No Drawing. Application December 27, 1952, SerialNo. 328,268

7 Claims.

This invention relates to an adrenocorticotrophin preparation suitablefor intramuscular or subcutaneous injection and having an enhancedadrenocorticotrophic effect.

The present application is a continuation-inpart of my prior copendingapplication Serial No. 179,391, now abandoned, filed August 14, 1950.

The adrenocorticotrophic hormone is a substance secreted by the anteriorlobe of the pituitary gland and presumably is carried by the bloodstream in the animal body to the adrenal gland where it exerts aninfluence on the development, growth, and activity of the adrenalcortex. In relatively recent years, extracts of the adrenocorticotrophichormone derived from the pituitary glands of animals such as hogs,sheep, and cattle have been found extremely effective in alleviatingcertain pathological conditions in human beings when injectedparenterally, and there has been an increasing demand for quantities ofthe hormone suitable for injection into the human body.

In view of the relatively short supply of animal pituitary glands, fromwhich the adrenocorticotrophic hormone is obtained, and in view of theextremely small size and content of the pituitary glands themselves, itis of importance that methods be developed not only for obtaining theutmost in yield of active substance from the available glands but alsofor increasing the potency oi the active substance to as great an extentas possible.

An object of the present invention is to provide an adrenocorticotrophinpreparation which is substantially more potent than the active extractfrom which the preparation is made. A further object of the invention isto provide a combination of adrenocorticotrophin and carrier which issafe and convenient for intramuscular or subcutaneous injection while atthe same time exhibiting a coaction or cooperation between the hormoneand carrier resulting in a substantial magnification ofadrenocorticotrophic effect. A still further object is to provide acombination of adrenocorticotrophin and carrier which may be combinedwith other substances so that the resulting preparation possesses notonly an enhanced adrenocorticotrophic eiiect but in addition along-lasting effect more nearly simulating the natural feeding orsecretion of the hormone ification proceeds.

In one phase of my invention, I introduce the adrenocorticotrophinsubstance in a gelatin vehicle. The adrenocorticotrophic hormoneemployed in combination with gelatin as the vehicle is significantlymore potent than the same adrenocorticotrophic hormone injected in anordinary aqueous solution. The magnification of eifect resulting fromthe use of gelatin has been found to be a doubling or tripling ofadrenocorticotrophic potency.

The above-described increase in effect is a true potentiation, asdistinguished from a mere retarding efiect which may sometimes be causedby the viscosity of the vehicle. This may be illustrated by the factthat a gelatin-adrenocorticotrophin preparation produces, immediatelyupon injection, a sudden strong response which is substantially inexcess of the response caused by an equal dose of the same substance inan aqueous solution from which the gelatin is omitted. I have foundthat, during the 12 to 16 hours following injection, the pattern ofresponse to a given dose of gelatin-adrenocorticotrophin is similar tothat of an identical dose of salineadrenocorticotrophin, except that thegelatinadrenocorticotrophin response is always stronger at anyparticular point of time, as if a much greater dosage had been given.This type of reaction cannot be explained merely on the basis ofabsorption retardation but it believed to be based on a unique propertyof the gelatin which is displayed when the gelatin is used incombination with the adrenocorticotrophic hormone but not when it isused with other biologically active substances. Studies on a greatnumber of patients have demonstrated that, by injectingadrenocorticotrophin in gelatin rather than in the usual salinesolution, the same general clinical response may be achieved by usingonly onehalf to one-third of the adrenocorticotrophin ordinarily used.

The gelatin employed in the practice of my invention may be anynon-antigenic gelatin suitable for parenteral injection. There arenumerous pharmaceutical gelatins on the market, each of which has itsown characteristic properties, such as source, isoelectric point, pH,viscosity, etc. However, I have found that any of the pharmaceuticalgelatins have the effect of increasing the potency of theadrenocorticotrophic hormone, and the only important factors governingsuitability for use are the purely practical matters involved in thetechniques of parenteral administration. For example, in theadministration of a drug contained in a vehicle, it is desirable thatthe vehicle be fluid, or at least easily liquefied, at room temperatureand that its gel pointbesuch. that it does' notsolidify in the injectionneedle orin the body tissue after injection. In the case of aqueousgelatin solutions which have too high a gel point 01' viscosity,

this may be accomplished by a partialliydrolysis or by decreasing thegelatin concentration,.- etc. Partial hydrolysis has a further advantagein that the autoclaving step by which hydrolysis'is-usual- 1yaccomplished is also eifectiveininsuring that the gelatin isnonantigenic. I prefer to use about a 16% aqueous solutionuofi apartiallyhydrolyzed pharmaceutical gelatin which is neutral;

or has an isoelectric point somewhat on the acid side. It is usuallydesirable that the gelatinconcentration be within the range of 5 to 35%by weight. of the-injection solutionpalthough, as mentioned", this i'iSimportant mostly: from the standpoint of the patients comfort-durmg orafter administration :of I the hormone, and the effectiveness of" thegelatin. in increasing a'dreno corticotrophiii: potency :ismanifested.to some degree even at: gelatinzconcentrations aboverandi below theselimits.

Any adrenocorticotrophic. substance may be used with gelatin to providethe improved adrenocorticotrophict effectiveness: contemplated bythepresent invention. The adrenocorticotrophic hormone has notzyetbeenisolated; and its molecular structurehas' not-as yet'been definitelycharacterized. I prefer to employa fairly well purified substance. such.as the acid hydrolyzed product obtained: byJoseph? D. Fisher in. his.copending: application Serial No; 122,588, wherein onecmethodof'preparingthe=product invol'ves preparing an acidr acetoneadrenocorticotrophin powder by known-means; removing the bulk, of: thecontaminants-by extracting: the acidacetone powder with. 'dibasi'c'.sodium' phosphate solution, precipitating the a'drenocorticotrophicsubstancefronr the'dibasic sodium' phosphate so-- lution byad'dingammoniumsulfate; centrifuging, resuspendingc the: precipitate indistilled water, dialyzing, adjusting. the"pI-I -tovabout"1'.3 withhydrochloric acid, boiling: at I00" C; for about'2 /z" hours, andthenzdial'yzing andIyophili'zing; or the pepsin hydrolyzed productdescribed by Lottie J. Walaszek in 'her: 'copen'ding': application-Serial No.'.l'.72;01l wherein onemethod which is described for preparingthe product involves hydrolyzing a relatively puread'renocorti'cotrophic' substance with pepsin; treating the pepsinhydrolyzed substance with trichloroacetic acid to precipitate inertmaterials, washing the unprecipitate'd" material with a solvent,'such asether, which dissolves the trichloroacetic' acid-butwhich does notdissolve the hormone substance, and dialyzing the solution; or thecellulose purified product obtained by Irby MJ'Bunding in his co pendingapplication Serial No. 13791650 wherein one method Wh'iChLiSdescribedrfor preparingthe product involves {suspendingacrelat'ively'purified adrenocorticotrohic substance; 1114a;SQl'lltiOIlLCUD.

taining finely-divided filter. paper (a .cellulose;

material) inorder. to absorb the: adrenocortico.--

trophin-om thepaper separating the solid phase.

-certainty at the present time. .s-ibleexplanation may be that thereare, in the tissues surrounding the site of injection of theadrenocorticotrophic hormone, certain agents which have aspecific-destructive force upon the adrenocorticotrophin, althoughapparently not upon other injected drugs. This possibility is.supportedlbyexperiments described in an article entitled Inactivationof Adrenocorticotrophic Hormone .in Vitro by Tissues by I. I. Geschwindand'CI H. Li (Endocrinology, vol. 50, No. 2, p. 226, February 1952),where it is stated that certain body tissues-*have been found to have aninactivating-*-efi'ect-on'ACTH; Inviewof this, it

appears that-when "the*adrenocorticotrophic hormone is injectedsubcutaneously or intramuscularly-, it i's expose'd to these destructiveforces in'the surrounding tissue, and therefore -a substantial portionof its activityis lost before being released into-the bloodstream fortransfer to the adrenal glands; However, gelatin, when used incon'ibination with the"adrenocorticotrophic hormone, appears to possessa'specific stabilizingeffect upon the hormone, thusprotecting it againstinactivation bythedestructive-forces in the tissue surrounding thesiteofinjection andconsequent ly enabling the="full activedose of hormone toreach the blood stream wherein it is carried safely: to the adrenals.

='In;another phase of my invention, I introduce theadrenocorticotrophin-gelatin product inassociationl with aretarding-agent compatible with gelatin; "thereby: obtaining thebenefits not only of increased potency-"but also of prolongedtherapeutici activity. 'As'the retarding agent, I may use: any of thewell -known-substanc es whi'ch reduce-the rate of 'absorption of thedrug in the blood stream either-by" virtue of the viscosity-orthe:astringent properties of these substances. For example, an: astringentsuch as aluminum phosphate maybe' employed. "Similarly, theretarding'agentmay bealuminum hydroxide, magnesium phosphate,an'd'related materials which are difiicultly water-soluble astringents;Other retardingragents, such as polyvinyl pyrrolidone, whichdependsxon-l its'viscous character for retarding'r'elease of' thehormone into the blood stream, may lie-used, or combinations of suchast'ringents and viscous vehicles have been found effective when usedinassociation with the adrenocorticotrophin gelatinproduct of thepresent invention.

More specifically, one embodiment of this,

latter phase of the invention may be carried out in the' followingmanner: The astringent material, sucl'i' as-alumirrum phosphate, forexample, iniiinea particulate form is prepared in distilled water, and.the suspension is added to the adrenocorticotrophicrhormone preparation.The

gelatinsolutioncis thenzadded and-the wholemixedithoroughly. Aftersterilization, thematerial: is, readyqf-or. injection. In'furtherspecific.

embodiments, polyvinyl pyrrolidone may either be substituted for thealuminum phosphate, or used in addition thereto.

The ratio of the astringent to the adrenocoi ticotrophic hormonesubstance may vary considerably depending upon the effects desired. Inthe case where aluminum phosphate is used as the astringent, forexample, I prefer to have at least one half as much aluminum phosphateas the adrenocorticotrophic hormone solids. It is undesirable to usemuch more than twice as much aluminum phosphate as the hormone substancebecause of local irritation. A preferred range is from one-half to twotimes as much aluminum phosphate as the hormone substance solids, and Iprefer to use an amount of aluminum phosphate equal to the amount of thehormone; for example, 25 mg. adrenocorticotrophic hormone substance for25 mg. aluminum phosphate. It will be understood, however, that suchratios may be varied considerably depending upon the medical effects tobe achieved.

Specific examples of the products of my inven tion and methods of theirpreparation are described as follows:

EXAIVIPLE I A lyophilized adrenocorticotrophin product prepared by themethod described in Fisher application Serial No. 122,588, wasreconstituted A lyophilized adrenocorticotrophin product was prepared asfollows: Crude pork pituitary glands, immediately after being removedfrom the animal, were frozen and kept in a frozen condition until theywereready to be processed. The glands were then ground with dry ice andthen, after adding a solution of an organic solvent, the frozen glandswere thawed as rapidly as possible. The thawed glands were extracted inan aqueous medium which contained acetone at a pH of about 1.5. Afterextraction, the meat was separated from the liquid in a centrifuge, andthe centrifugate was treated with additional acetone to bring thepercentage of acetone to about 90%, at which point precipitationoccurred. The precipitate was separated from the liquor, dissolved inwater, and subsequently dried. The powder thus obtained was extractedwith a 0.1 N solution of dibasic sodium phosphate and the materialseparated in a centrifuge. The supernatant material remaining afterseparation was one-half saturated with ammonium sulphate to form aprecipitate and was again centrifuged. The precipitate was dialyzed forthree days in the cold to remove the ammonium sulphate. In this step theactive substance does not dialyze out. The active material was thenlyophilized.

The above lyophilized product, which is of the type sometimes referredto as whole protein adrenocorticotrophin, and which displays aninability to dialyze through an ordinary dialysis membrane, wasreconstituted with distilled water, and one volume of this was added toone volume of a solution containing 32% by weight of a partiallyhydrolyzed gelatin and 1% phenol.

The resulting combined solution, containing 16% gelatin and 0.5% phenolin addition to the adrenocorticotrophin, was subjected to standard assayand was found to be from 2 to 3 times as potent as the originaladrenocorticotrophin administered in ordinary saline.

EXAMPLE III A quantity of adrenocorticotrophin was prepared byextracting acetone dehydrated anterior pituitaries of hogs with glacialacetic acid at 70 C. adding ,4; volume of acetone and a small portion ofNaCl to the solution precipitate unwanted materials; adding an equalvolume of ethyl ether to the solution to precipitate a crudeadrenocorticotrophin; purifying the precipitate by adsorption onoxycellulose and elution therefrom; and adding an anion exchange resinto the eluate to convert the adrenocorticotrophin to the acetate.

The acetate solution thus formed contained adrenocorticotrophin in theso-called polypeptide form and a portion of this was diluted one volumeto one volume with a solution containing 32% gelatin and 1% phenol toproduce a combined solu tion containing 16% gelatin and /2% phenol. Thiswas assayed by the standard U. S. P. method involving the adrenalascorbic acid depletion test on hypophysectomized rats and also bystandard clinical methods and was found to have a potency at least twicethat of the same adrenocorticotrophin substance assayed in ordinaryaqueous vehicle containing A;% phenol but no gelatin.

EXAMPLE IV Preparation of adrenoco'rticotrophin aluminum phosphate ingelatin. 25 mg. adrenocortz'cotrophine per cc. (in terms of Standard)Gelatin (non-antigenic) grams 72.0 Dextrose (anhydrous) do 32.0 Aceticacid cc 2.0 Distilled water cc 130.0

thoroughly by warming andshaking the finished preparation.

5. The product was filled into 10 cc. vials and: capped with closuressuitable to allow withdrawal Toxicityin guinea -pigs No. toxic symptoms.

Sterility .Sterile.

Adrenocorticotrophin po- 25 mg.

tency by adrenal ascorbic per cc. acid depletion in hypophysectomizedrats.

Standard 8,.The product was labeled, packaged and tested clinically.

EXAMPLEV Preparation of adrenocorticotrophin aluminum "phosphate ingelatin 1. Prepared the following:

Gelatin (non-antigenic) grams 60.0 Procaine hydrochloride do 5.0Chlorobutanol 'do 1.0 Trisodium phosphate do 2.0 Distilled water cc166.0

The ingredients were mixed and melted on a water bath. The resultingsolution was clarified by subjecting it to a vacuum to remove airbubbles.

2. Prepared washed AlPOi suspension 20 mg. per cc. as in step 1, ExampleIV.

,3, Placed 1.0 gram adrenocorticotrophin (potency 350177 per cent ofStandard) into a suitable container. Added 25 cc. aluminum phosphatesuspension (prepared in step 2). Allowed to stand thirty minutes. Thenadded '75 cc. of the preparation made in step 1. The preparation wasmixed thoroughly with the aid of heat and agitation.

4. The finished bulk lot was filled into 10 cc. vials and sealed withrubber closures.

5. The vials containing the product were autoclaved at 'lb. pressure forthirty minutes to sterilize the contents.

6. Sampleswere subjected to control tests with the indicated results:

Standard .7. The product was labeled, packaged and tested clinically.

EXAMPLE VI 1. Prepare a solution of adrenocorticotrophin in water about1 per cent concentration.

2. Prepare an aqueous colloidal suspension of aluminum phosphate to..50mg./cc.

.3. Add the preparation of Step2 to the preparation of step 1 to providea 10 to mg. AlPO4 for each 25 mg. adrenocorticotrophin (Standardpotency). Mix and let stand one hour. Lyophilize.

4. Prepare a solution of gelatin, .7 /z%, and polyvinyl pyrrolidone,10%, in water.

. 5.. Add the preparation of. step. 4 above to .the preparation of step3 above, with trituration,,.to produceaa -col-loidal suspensioncontainin desired concentration of.adrenocorticotrophin.

6. Vial :the product and. sterilize by means of heat.

EXAIVIPLE VII 1. Weigh a suitable quantity of adrenocorticotrophin.

2. Prepare a solution of l /2% gelatin and 10% polyvinyl pyrrolidone inwater.

-3. Add the preparation of step 2 above to the preparation of .step 1above, with trituration, to prov-idethe desired concentration ofadrenocorticotrophin. Adjust pH to 3.0 to 4.0 to provide clear .ornearly clear solution.

4. Vial the product and sterilize by means of heat.

Whenever the word Standard is referred to herein, 1'. mean theInternational Standard which has been adopted'by the World HealthOrganization. One unit under the International Standard is defined .asequivalent to the potency of 1 mg. of preparation LA-l-A when tested bythe method of Sayers, G. Sayers, and L. A. Woodbury, Endocrinology, 42,379 (1948).

While in the foregoing specification specific embodiments of thisinvention have been set out in considerable detail for the purpose ofillustration, it will be apparent to those skilled in the art that manyof the details set forth can be variedwidely without departing from thespirit of the invention.

I claim:

1. An adrenocorticotrophin preparation suitable ior parenteraladministration, comprising adrenocorticotrophin substance andnon-antigenie-pharmaceutical gelatin.

2. An adrenocorticotrophin preparation suitable for parenteraladministration, comprising adrenocorticotrophine substance and a 16%aqueous solution of non-antigenic pharmaceutical gelatin.

3. An adrenocorticotrophin preparation suitable for parenteraladministration, comprising adrenocorticotrophin substance, non-antigenicpharmaceutical gelatin, and polyvinyl pyrrolidone.

1. An adrenocorticotrophin preparation suitable for parenteraladministration, comprising adrenocorticotrophin substance, non-antigenicpharmaceutical gelatin, and a non-toxic dinicultly Water-solubleastringent selected from the group consistin of aluminum phosphate,aluminum hydroxide, and magnesium phosphate.

5. An adrenocorticotrophin preparation suitable for parenteraladministration, comprising adrenocorticotrophin substance, non-antigenicpharmaceutical gelatin, and aluminum phosphate.

6. An adrenocorticotrophin preparation suitable for parenteraladministration, comprising adrenocorticotrophin substance, non-antigenicpharmaceutical gelatin, polyvinyl pyrrolidone, anda non-toxicdiflicultly water-soluble astringent selected from the group consistingof aluminum phosphate, aluminum hydroxide, and magnesium phosphate.

7. An. adrenocorticotrophin preparation suitable ior parenteraladministration, comprising adrenocorticotrophin substance, non-antigenicpharmaceutical gelatin, polyvinyl pyrrolidone, and aluminum phosphate.

ROBERT E. THOMPSON.

(Re er ces onioi g Pa e) References Cited in the file of this patentUNITED STATES PATENTS Number Number Name Date Lautenschlager May 31,1932 Friedrich Feb. 13, 1940 Sevag May 28, 1940 Junkmann Dec. 28, 1943Saunders Dec. 31, 1946 Abramson Feb. 11, 1947 Durel June 28, 1949 KoreaNov. 15, 1949 Welch Dec. 20, 1949 FOREIGN PATENTS Country DateSwitzerland June 16, 1943 OTHER REFERENCES Recent Progress in HormoneResearch, vol.

II, pages 1-73 (pages 5, 37-46 and 73 are especially pertinent); pub.1952 by Academic Press Inc.

Hayashida et a1., Endocrinology, Vol. 50, No. 2, Feb. 1952, pages187-191. (Copy in Pat. Off. Sci. Libr.)

Bates, Endocrinology, vol. 52, Mar. 1953, pages 266-271.

Carlinfanti in Mfg. Chemist, Apr. 1949, vol. 20, page 157.

Pincus, The Hormones, vol. I, 1948, pages 665-673.

Unlisted Drugs, v01. 2, Aug. 31, 1950, No. 8, page 85.

Endocrinology, v01. 50, Feb. 1952, pages 274-276, article by Farrell eta1.

Schafers Essentials of Histology, pub. 1949. 15th ed., pages 96 and280-288.

1. AN ADRENOCORTICOTROPHIN PREPARATION SUITABLE FOR PARENTERALADMINISTRATION, COMPRISING ADRENOCORTICOTROPHIN SUBSTANCE ANDNON-ANTIGENIC PHARMACEUTICAL GELATIN.